The present invention relates to transcriptional gene silencing (TGS) in mammalian, including human, cells that is mediated by small interfering RNA (siRNA) molecules. The present invention also relates to a method for directing histone and/or DNA methylation in mammalian, including human, cells. It has been found that siRNAs can be used to direct methylation of DNA in mammalian, including human, cells.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
RNA interference (RNAi) is a process in which double stranded RNA (ds RNA) induces the postranscriptional degradation of homologous transcripts, and has been observed in a variety of organisms including plants, fungi, insects, protozans, and mammals (Moss et al., 2001; Bernstein et al., 2001; Elbashir, et al., 2001a, 2001b). RNAi is initiated by exposing cells to dsRNA either via transfection or endogenous expression. Double-stranded RNAs are processed into 21 to 23 nucleotide (nt) fragments known as siRNA (small interfering RNAs) (Elbashir et al., 2001a, 2001b). These siRNAs form a complex known as the RNA Induced Silencing Complex or RISC (Bernstein et al., 2001; Hammond et al. 2001), which functions in homologous target RNA destruction. In mammalian systems, the sequence specific RNAi effect can be observed by introduction of siRNAs either via transfection or endogenous expression of 21-23 base transcripts or longer hairpin precursors. Use of siRNAs evades the dsRNA induced interferon and PKR pathways that lead to non-specific inhibition of gene expression. (Elbashir et al., 2001a).
The discovery of siRNAs permitted RNAi to be used as an experimental tool in higher eukaryotes. Typically, siRNAs are chemically synthesized as 21 mers with a central 19 by duplex region and symmetric 2-base 3′-overhangs on the termini. These duplexes are transfected into cells lines, directly mimicking the products made by Dicer in vivo. Most siRNA sequences can be administered to cultured cells or to animals without eliciting an interferon response (Heidel et al., 2004; Ma et al., 2005; Judge et al., 2005). There are some reports that particular motifs can induce such a response when delivered via lipids (Judge et al., 2005; Sledz et al., 2003; Hornung eta 1, 2005), although a cyclodextrin-containing polycation system has been shown to deliver siRNA containing one such putative immunostimulatory motif that achieves target gene down-regulation in mice without triggering an interferon response (Hu-Lieskovan et al., 2005), even in a disseminated tumor model.
It has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21 mers at the same location. At the site most extensively examined in this study, EGFPS1, only minor differences in potency were seen between duplexes with blunt, 3′-overhang or 5′-overhang ends, and a blunt 27 mer duplex was most potent (Kim et al., 2005). Increased potency has similarly been described for 29 mer stem short hairpin RNAs (shRNAs) when compared with 19 mer stem hairpins (Siolas et al., 2005). While the primary function of Dicer is generally thought to be cleavage of long substrate dsRNAs into short siRNA products, Dicer also introduces the cleaved siRNA duplexes into nascent RISC in Drosophila (Lee et al., 2004); Pham et al., 2004; Tomari et al., 2004). Dicer is involved in RISC assembly and is itself part of the pre-RISC complex (Sontheimer et al., 2005). The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27 mer) instead of a product (21 mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.
Not all 27 mers show this kind of increased potency. It is well known that shifting a 21 mer siRNA by a few bases along the mRNA sequence can change its potency by 10-fold or more (Holen et al., 2002); Harborth et al., 2003; Reynolds et al., 2004). Different products that result from dicing can have different functional potency, and control of the dicing reaction may be necessary to best utilize Dicer—substrate RNAs in RNAi. The EGFPS1 blunt 27 mer studied in Kim et al. (2005) is diced into two distinct 21 mers. Vermeulen and colleagues reported studies where synthetic 61 mer duplex RNAs were digested using recombinant human Dicer in vitro and examined for cut sites using a 32P-end-labeled gel assay system. Heterogeneous cleavage patterns were observed and the presence of blunt versus 3′-overhang ends altered precise cleavage sites (Vermeulen et al., 2005). Dicing patterns were studied at a variety of sites using different duplex designs to see if cleavage products could be predicted. It has been found that a wide variety of dicing patterns can result from blunt 27 mer duplexes. An asymmetric duplex having a single 2-base 3′-overhang generally has a more predictable and limited dicing pattern where a major cleavage site is located 21-22 bases from the overhang. Including DNA residues at the 3′ end of the blunt side of an asymmetric duplex further limits heterogeneity in dicing patterns and makes it possible to design 27 mer duplexes that result in predictable products after dicing.
It has been found that position of the 3′-overhang influences potency and asymmetric duplexes having a 3′-overhang on the antisense strand are generally more potent than those with the 3′-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript. Novel designs described here that incorporate a combination of asymmetric 3′-overhang with DNA residues in the blunt end offer a reliable approach to design Dicer—substrate RNA duplexes for use in RNAi applications. See also U.S. published application Nos. 2005/0244858 A1 and 2005/0277610 A1, each incorporated herein by reference.
Recently, several groups have demonstrated that siRNAs can be effectively transcribed by Pol III promoters in human cells and elicit target specific mRNA degradation. (Lee et al., 2002; Miyagishi et al., 2002; Paul et al., 2002; Brummelkamp et al., 2002; Ketting et al., 2001). These siRNA encoding genes have been transiently transfected into human cells using plasmid or episomal viral backbones for delivery. Transient siRNA expression can be useful for rapid phenotypic determinations preliminary to making constructs designed to obtain long term siRNA expression. Of particular interest is the fact that not all sites along a given mRNA are equally sensitive to siRNA mediated downregulation. (Elbashir et al., 2001a; Lee et al., 2001; Yu et al., 2002; Holen et al., 2002).
In contrast to post-transcriptional silencing involving degradation of mRNA by short siRNAs, the use of long siRNAs to methylate DNA has been shown to provide an alternate means of gene silencing in plants. (Hamilton et al., 2002). In higher order eukaryotes, DNA is methylated at cytosines located 5′ to guanosine in the CpG dinucleotide. This modification has important regulatory effects on gene expression, especially when involving CpG-rich areas known as CpG islands, located in the promoter regions of many genes. While almost all gene-associated islands are protected from methylation on autosomal chromosomes, extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X-chromosomes of females. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers. In this last situation, promoter region hypermethylation stands as an alternative to coding region mutations in eliminating tumor suppression gene function. (Herman et al., 1996).
U.S. published application No. 2004/0096843 A1, incorporated herein by reference, is directed to methods for producing double-stranded, interfering RNA molecules in mammalian cells. These methods overcome prior limitations to the use of siRNA as a therapeutic agent in vertebrate cells, including the need for short, highly defined RNAs to be delivered to target cells other than through the use of synthetic, duplexed RNAs delivered exogenously to cells. U.S. published application No. 2004/0091918 A1, incorporated herein by reference, is directed to methods and kits for synthesis of siRNA expression kits.
Small interfering RNA (siRNA) mediated transcriptional gene silencing (TGS) was first observed in doubly transformed tobacco plants which exhibited a suppressed phenotype of the transformed transgene. Careful analysis indicated that methylation of the targeted gene was involved in the suppression (Matzke et al., 1989). TGS mediated by dsRNAs was further substantiated in plants infected with a cytoplasmic dsRNA virus; nuclear transgenes with promoters homologous to sequences in the virus were found to be silenced (Wassenegger et al., 1994; Wassenegger, 2000). siRNAs that target promoter sequences have also been shown to cause TGS in the yeast S. pombe and in Drosophila (Pal-Bhadra et al., 2002; Schramke and Allshire, 2003). Transcriptional silencing by siRNA most likely functions as a genome defense mechanisms that target chromatin modifications to endogenous silent loci such as transposons and repeated sequences (Seitz et al., 2003; Soifer et al., 2005). In plants and yeast siRNA-induced silencing is accompanied by DNA methylation of homologous sequences, de novo DNA methylation in Arabidopsis thaliana requires siRNA metabolizing factors, and maintenance of S. pombe centromeric heterochromatin depends on siRNA-directed histone H3 lysine 9 methylation (Chan et al., 2004; Jones et al., 2001; Mette et al., 2000; Volpe et al., 2002; Zilberman et al., 2003).
While dsRNAs induce sequence-specific methylation of DNA in plants and yeast, regulating gene expression at the transcriptional level, it was not known until recently how applicable this phenomenon was in mammalian cells. Recent reports have documented that siRNAs targeted to 2 different genes, specifically the promoter regions, can induce transcriptional silencing via histone and DNA methylation in human cells (Morris et al., 2004b; Kawasaki and Taira, 2004; Kawasaki et al., 2005). While these reports were intriguing many questions regarding the underlying mechanism remained.
It is desired to utilize this activity and to use siRNAs to induce transcriptional gene silencing in cells.